OIL-IN-WATER NANOEMULSIONS 335 from Spec-Chem Ind (Zhongshan, China). Butylated hydroxytoluene, xanthan gum, and beeswax were purchased from Fluka (St. Louis, USA). PEG-40 hydrogenated castor oil and phenonip were provided by Sime Darby Research Sdn. Bhd (Selangor, Malaysia). Deionized water was produced by using deionized water system (Milli-Q System Massachusetts, USA). Two surfactants used were polyoxyethylene sorbitan monooleate (Tween 80) and sorbitan monooleate (Span 80), purchased from Sigma Aldrich (St. Louis, USA). Compositions of formulation are shown in Table I. EMULSION PREPARATIONS Rotor–stator method. The samples were produced by emulsifi cation using hot–hot process. Emulsions were prepared where both oil and aqueous phases were separately warmed up to 70 ± 5°C. Xanthan gum was dispersed in deionized water at 0.8% w/w. Preparation of oil phase was performed by homogenizing 5% w/w of mixed Tween 80 and Span 80 (4:1) into oil phase of mixture with a Polytron homogenizer (Kinematica GmbH, Lucerne, Switzerland) rotor–stator. An emulsion sample of 100-ml total volume was prepared by pouring the oil phase into the aqueous phase and homogenizing at 6000 rpm for 5 min. The temperature was lowered to 40°C when the active ingredients and preservative were added. The emulsions were then subjected to mixing using stirrer (Ika-Werke, Staufen, Germany) at 200 rpm until reaching room temperature (25 ± 2°C) for 4 h. Ultrasonic cavitation method. As a comparison, emulsions were also prepared using UP400S Hielscher Sonifi er (Teltow, Germany) of 400 W nominal power and a fre- quency of 24 kHz equipped with a 22-mm sonotrode tip. This was placed in a custom- built cooling jacket. Chilled water at 3°C was continuously passed through this jacket. Emulsions were prepared where both oil and aqueous phases were separately warmed up to 70 ± 5°C. Xanthan gum was dispersed in deionized water at 0.8% w/w. An emul- sion sample of 100-ml total volume was prepared and prehomogenized at 6000 rpm for 5 min with a Polytron homogenizer (Kinematica GmbH) rotor–stator. The tempera- ture was lowered to 40°C. At 40°C, the active ingredients and preservative were added. Table I Chemical Compositions of Formulation Prepared By Rotor–Stator Homogenizer and Ultrasonic Cavitation Ingredients wt% Function External water phase (75°C ± 5°C) Water, deionized 63.70 Diluent/solvent Xanthan gum 0.80 Thickener, emulsion stabilizer Internal oil phase (75°C ± 5°C) Palm oil esters 15.80 Skin conditioning, emulsifi er PEG-40 hydrogenated castor oil 10.00 Surfactant/emulsifi er Beeswax 0.50 Skin conditioner Polyoxyethylene sorbitan monooleate 4.00 Surfactant/emulsifi er Sorbitan monooleate 1.00 Surfactant/emulsifi er Active ingredients phase (40°C ± 5°C) Butylated hydroxytoluene 0.10 Lipophilic antioxidant Tocotrienol 2.90 Vitamin E derivative—antioxidant Magnesium ascorbyl phosphate 0.50 Vitamin C derivative—antioxidant Phenonip 0.70 Preservatives

JOURNAL OF COSMETIC SCIENCE 336 The emulsions were further homogenized using ultrasonic cavitation for 5 min. The sonifi er tip horn was adjusted to 2 cm below the surface of a 100-ml sample. Sonication was performed at amplitude of 30 μm and 0.5 cycles. PHYSICAL MEASUREMENTS A Nanophox (Sympatec GmbH Instruments, Clausthal-Zellerfeld, Germany) was used to analyze the particle size distribution (PSD) of the fi ner emulsions formed. This device performs size measurements by photon cross-correlation spectroscopy, which measures the Brownian motion of colloidal particles, thus allowing the determination of their PSD. Samples were diluted with double-distilled water at one part of sample to two parts of water. The average drop size, expressed as the Sauter mean diameter (d32 = Σ nid3i/Σ nid2i, representing a surface average value), and the drop size distribution were obtained by means of a laser diffractometer according to the Mie theory. The Mie theory is a rigorous solution for the scattering intensity from a spherical, homogeneous, isotropic, and non- magnetic particle of any diameter in a nonabsorbing medium. A refractive index of 1.460 was used for palm oil esters in Mie theory calculations. Emulsion particle size results are an average of six measurements of freshly prepared emulsions. Zeta potential was measured using a Zetasizer Nano (Malvern Instruments, Worcester- shire, UK). The samples were diluted (1:200) with distilled water and added into the equipment chamber. To obtain stable nanoemulsions (no fl occulation and coalescence of the nanodroplets), zeta potential should usually reach a value of ±30 mV. The zeta poten- tial results were obtained by applying the Henry equation: 2H]f 3K E ka U (1) where UE is the electrophoretic mobility, ε is the dielectric constant, ζ is the zeta poten- tial, η is the viscosity of the continuous phase, and f(ka) is the Henry’s function (13). The rheology of O/W emulsions was characterized by using Kinexus Rotational Rheom- eter (Malvern Instruments, Worcestershire, UK). Two different rheological measure- ments were made to characterize the emulsions. First, viscosity versus shear stress, viscosity versus shear rate, and viscosity versus time plot at elevated temperature were applied to the emulsion samples. Second, oscillatory rheological measurements were made in the linear viscoelastic region, using 4°/40 mm cone and plate geometry and gap of 0.100 mm. All measurements were carried out at room temperature of 25.0 ± 0.5°C. RESULTS AND DISCUSSION SIZE DISTRIBUTION STUDIES The size of an emulsion droplet formed by homogenization is controlled by the interplay between droplet breakup and droplet coalescence (14). Droplet breakup is controlled by the type and amount of shear applied to droplets, and the droplet resistance to deforma- tion is determined by the surfactant. Droplet coalescence is determined by the ability of the surfactant to rapidly adsorb into the surface of newly formed droplets (14). In the present study, Tween 80 and Span 80 were used as the mixed surfactants and the effect of PSD for emulsions prepared by rotor–stator emulsifi cation and ultrasonic